1. Field of the Invention
This invention relates to the field of diagnostic assays, and in particular to the determination of platelet function activity on blood samples to study effects of anti-platelet compositions, and more particularly the dual use of a platelet activator (like ADP) and a platelet inhibitor (like Prostaglandin E1 (PGE1)) for measurement of platelet function of P2Y12 inhibitors, including clopidogrel, CS-747 (Sankyo), and ticlopidine with increased sensitivity.
2. Description of the Related Art
The role of platelets in mammalian physiology is extraordinarily diverse, but their primary role is in promoting hemostasis. In many situations, an evaluation of the ability of blood to clot is desired, a parameter that is frequently controlled by the ability of platelets to adhere and/or aggregate. Of interest, therefore, is the assessment of the adhesive functions of platelets. For example, questions of interest include whether to administer drugs that will block, or promote, clot formation, or whether to detect deficiencies in platelet function prior to surgical procedures. Also of interest is evaluating the effectiveness of a platelet inhibitor that is being tested as a new drug or is being used as approved clinical treatment in a patient.
Platelets are known to aggregate under a variety of conditions and in the presence of a number of different reagents. Platelet aggregation is a term used to describe the binding of platelets to one another. Platelet aggregation in vitro depends upon the ability of platelets to bind fibrinogen to their surfaces after activation by an aggregation-inducing agent such as ADP or collagen.
Platelets play a critical role in the maintenance of normal hemostasis. When exposed to a damaged blood vessel, platelets will adhere to exposed sub-endothelial matrix. Following the initial adhesion, various factors released or produced at the site of injury such as thrombin, ADP and collagen activate the platelets. Once platelets are activated, a conformational change occurs in the platelet glycoprotein GPIIb/IIIa receptor, allowing it to bind fibrinogen and/or von Willebrand factor.
It is this binding of the multivalent fibrinogen and/or von Willebrand factor molecules by GPIIb/IIIa receptors on adjacent platelets that results in the recruitment of additional platelets to the site of injury and their aggregation to form a hemostatic plug or thrombus.
In vitro platelet aggregometry is the laboratory method used to assess the in vivo ability of platelets to form the aggregates leading to a primary hemostatic plug. In this technique an aggregating agent such as ADP or collagen is added to whole blood or platelet-rich plasma and aggregation of platelets monitored. Platelet aggregometry is a diagnostic tool that can aide in patient diagnosis and selection of therapy. Current assays to measure platelet aggregation are expensive, time-consuming, cumbersome, and generally not suitable for a clinical environment.
A rapid platelet function assay has recently been developed and is described in U.S. Pat. No. 5,763,199 (Coller). The assay determines glycoprotein (GP)IIb/IIIa receptor blockade in whole blood. Agglutination of small polymeric beads coated with a GPIIb/IIIa ligand such as fibrinogen results when the beads are contacted with whole blood containing platelets with activated GPIIb/IIIa receptors that are not blocked. Failure to agglutinate indicates either failure of the GPIIb/IIIa receptors to become activated and/or blockade of the GPIIb/IIIa receptors. In a preferred embodiment, the addition of a thrombin receptor activator results in an assay that is rapid and convenient enough to be performed at the bedside and that results in agglutination of the small polymeric beads within a convenient, known period of time if the GPIIb/IIIa receptors are not blocked. The assay includes the ability to transfer blood to be tested from a collection container to an assay device without opening the collection container. This platelet aggregation assay can be conducted at the same time as the activated clotting time (ACT), which is performed to assess the adequacy of heparinization.
Platelet aggregation plays a key role in the pathogenesis of thrombosis and acute coronary artery disease. Evidence suggests that significant platelet function variability exists in the response to various antiplatelet agents. It has also been demonstrated that an inter-individual variability in platelet aggregation exists when P2Y12 antagonists such as clopidogrel are used for treatment of patients to achieve an anti-aggregation effect. The results of one study demonstrated that at least 10% of patients receiving the drug did not achieve the expected platelet aggregation inhibition (Muller I, Besta F, Schulz C, Massberg S, Schonig A, Gawaz M; Prevalence of clopidogrel non-responders among patients with stable angina pectoris scheduled for elective coronary stent placemen Thromb Haemost. 2003 May, 89(5):783-7).
Clopidogrel and ticlopidine are thienopyridine derivatives that inhibit platelet aggregation. They are believed to inhibit the binding of adenosine-5-diphosphate (ADP) to one of its receptors, the P2Y12 receptor. The pharmacological activity of clopidogrel is very similar to the pharmacological activity of ticlopidine. However, clopidogrel has been shown to have fewer side-effects than ticlopidine. Based on mounting evidence of the efficacy of clopidogrel in thrombotic disease, the use of clopidogrel and other P2Y12 antagonists are likely to increase significantly.
Since many patients with cardiovascular disease are currently taking one of the thienopyridine agents, a method for detection of resistance to a thienopyridine and assessment of the efficacy of thienopyridine treatment would be beneficial. Thus, there is a need to develop an assay that would provide information about aspirin and thienopyridine, e.g., clopidogrel or ticlopidine, sensitivity and efficacy of treatment in a given patient.
The effects of these agents on platelet function have been assessed with platelet aggregometry using ADP, collagen or other platelet activators. However, since ADP activates at least two different receptors (P2Y1 and P2Y12 and perhaps P2X1), it has the potential for lower specificity and background noise. Collagen is another choice. However collagen is highly variable due its quaternary structure, which dramatically affects it ability to activate platelets and due to the fact it is derived from biological tissue and sensitive to minor changes in temperature and pH. Neither collagen nor ADP provide specificity to the P2Y12 receptor and therefore by themselves are not the optimal choice for the determination of the effects of P2Y12 inhibitors. In particular as has been shown in several studies, the choice of concentration of these two agonists has significant effect on the degree of inhibition to P2Y12 antagonists that is measured.
Prostaglandins (PGs) belong to a ubiquitous class of chemicals known as eicosanoids. They are found in virtually every tissue in the body and have a very wide spectrum of biological activities. Eicosanoids are derivatives of arachidonic acid, a polyunsaturated fatty acid. The term eicosanoids includes the family of prostaglandins (PGs), prostacyclin, thromboxanes, and leukotrienes. The PGs are divided in different families depending on their structure, each designated by a letter (A, E, F, G, H, or I). In addition to this letter, each individual prostaglandin carries a digit that indicates the number of double bonds in its fatty acid side chain. For example, prostaglandin E1 (PGE1) belongs to the E family and has only one double bond in its side chain. PGs plays an important role in platelet aggregation and hemostasis (blood clotting) and typically have a marked vasodilator effect.
PGE1 is the theoretical cyclooxygenase metabolite of dihomo-γ-linolenic acid (DGLA), but it is virtually undetectable in the plasma of normal humans or other animals. Its pharmacology includes vasodilation, hypotension, and anti-platelet activities. PGE1 has been shown to inhibit platelet aggregation by increasing cyclic adenosine monophosphate (cAMP) concentrations within platelets. A number of groups have shown that the IC50 of PGE1 for the inhibition of ADP-induced human platelet aggregation is around 40 nM.